Summary of Work:Research this past year focused on the molecular characterization of glutamate receptors and their function in the auditory system. Four families of ionotropic glutamate receptors and eight metabotropic glutamate receptors have now been identified. To characterize the biochemical and cell biological properties of this complex set of proteins, we have developed selective antibodies to most of the ionotropic receptor subunits and to a number of the metabotropic receptors. This past year an antibody selective for the AMPA receptor subunit, GluR2, a key subunit in the regulation of calcium flux through the channel, was developed in our laboratory. Several studies addressed the biochemical and cell biological properties of glutamate receptors in the central nervous system and in the cochlea. Most neurons express multiple glutamate receptors and several subunits of any ionotropic receptor, and several studies addressed the role of these multiple receptors. Our studies of the cerebellar Purkinje neuron show that several glutamate receptors are co-expressed at its two synaptic populations, the climbing fiber input and the parallel fiber input. However, the delta subunit is only found postsynaptic of the parallel fiber input in the adult, but associated with both synaptic populations in the young animal. Glutamate receptor distribution was also studied in fusiform cells of the dorsal cochlear nucleus. This neuron receives two different glutamatergic inputs on it's apical and basal dendrites. Our studies show that different glutamate receptors are expressed postsynaptic of the two inputs. GluR4 and mGluR1a are found postsynaptic of inputs to basal dendrites, but not to those of apical dendrite synapses. These results establish, for the first time, that glutamate receptors are selectively targeted to synaptic populations on neurons. Furthermore, they indicate that two distinct AMPA receptor complexes are present in these cells, those with GluR4 and those lacking GluR4. In the cochlea we found that the delta 1 receptor is abundantly expressed in inner hair cells, but not in outer hair cells, suggesting a function, perhaps as an autoreceptor, on these cells. Inner hair cells do not receive glutamatergic input. The expression of the nicotinic receptor subunit, alpha 9, was characterized in the organ of Corti. Expression was found in both inner and outer hair cells, with proteins concentrated at the bases of both cell types. The promoter region of the alpha 9 receptor in mouse was isolated.